The goal of the proposed research is to characterize at the molecular level how mutations can alter the structure and substrate specificity of the clinically important TEM-1 beta-lactamase and also to develop new inhibitors of the enzymes. Specific objectives include: 1. Identify the amino acid residues that control beta-lactamase substrate specificity. Every amino acid position in the TEM-1 beta-lactamase has been randomized to sample all possible amino acid substitutions. All of the random substitutions will be screened to identify those amino acid substitutions that increase the catalytic activity of the enzyme for a number of beta-lactam antibiotics. In addition, mosaic enzymes between TEM-1 and the class A Mycobacterium fortuitum beta-lactamase will be constructed to identify the residues that are responsible for the large difference in substrate profile between these enzymes. 2. Determine the mechanism by which amino acid substitutions alter beta-lactamase structure and substrate specificity. Biochemical methods will be used to further characterize the catalytic properties of the specificity mutants. These studies will include purification of the mutant enzymes and determination of kinetic parameters. In addition, the mechanism of action of a set of substitutions in an active-site loop that were previously shown to alter beta- lactamase substrate specificity will be investigated by genetic and biochemical approaches. 3. Use combinatorial library technology to develop new inhibitors of beta-lactamase. Random peptide phage display technology will be used to develop new TEM-1 beta- lactamase inhibitors. In addition to providing an important research tool, these inhibitors may be useful in antibiotic therapy or may serve as lead compounds for the design of new inhibitors of beta-lactamase. In total, these studies should contribute to the long term goal of understanding how beta-lactamase will respond to the selective pressure of antibiotic therapy with the hope that such knowledge will lead to the design of new drugs and inhibitors that are immune to this response.